Tumor Suppressor Gene

ABSTRACT

A full-length cDNA encoding novel proteins involved in the control of cell proliferation (human Gros1-L and S) was successfully isolated from the human testis cDNA libraries. A full-length cDNA encoding the mouse homologues of the human Gros1 (mouse Gros1-L and S) was also isolated. The colony forming activity of cells exogenously expressing Gros1-L was significantly reduced, while that of cells expressing Gros1 antisense RNA was significantly increased.

This application is a divisional of application Ser. No. 12/888,572,filed on Sep. 23, 2010, which is a divisional of application Ser. No.12/626,290, filed on Nov. 25, 2009, issued as U.S. Pat. No. 7,825,220,which is a divisional of application Ser. No. 11/603,619, filed Nov. 22,2006, issued as U.S. Pat. No. 7,642,070, which is a divisional ofapplication Ser. No. 10/045,815, filed Oct. 26, 2001, issued as U.S.Pat. No. 7,153,935, which is a continuation-in-part of PCT ApplicationSerial No. PCT/JP00/02731, filed Apr. 26, 2000, and claims priority toJapanese Patent Application No. 11-118806, filed Apr. 26, 1999. Thedisclosure of these prior applications is incorporated by referenceherein.

TECHNICAL FIELD

The present invention relates to the field of biological science, morespecifically to the field of cancer research. In particular, the presentinvention relates to novel proteins involved in the proliferationmechanism of cells. The proteins of the present invention can be used,for example, as target molecules for developing drugs against cancer.

BACKGROUND

From a cytogenetic and molecular biology perspective, there appears tobe a non-random mutation on human chromosome 1p in many malignant tumors(Caron, Med. Pediatr. Oncol., 24:215-221, 1995; Schwab et al., GenesChromosomes Cancer, 16:211-229, 1996). For example, deletions in theregion of chromosome 1p have been found in various oncocytes(neuroblastomas [White et al., Eur. J. Cancer, 33:1957-1961, 1997,Gros16; Ariyama et al., Genomics, 25:114-123, 1995; Cheng et al.,Oncogene, 10:291-297, 1995], meningiomas [Ishino, et al., Cancer,83:360-366, 1998], pheochromocytomas, medullary thyroid carcinomas,neuroendocrine tumors [Moley et al., Cancer Res., 52:770-774, 1992], Tcell acute lymphoblastic leukemia (T-ALL) [Iolascon et al., Leukemia,11:359-363, 1997], colorectal cancers [Praml et al., Oncogene,11:1357-1362, 1995, Gros13; Bomme et al., Genes Chromosomes Cancer,21:185-194, 1998; Di Vinci et al., Cancer, 83: 415-422, 1998],mesotheliomas [Lee et al., Cancer Res., 56: 4297-4301, 1996], hepatomas[Chen et al., Cancer Genet Cytogenet, 86:102-106, 1996], endometrialcarcinomas [Arlt et al., Hum. Mol. Genet, 5:1017-1021, 1996], and breastcancers [Nagai et al., Cancer Res., 55:1752-1757, 1995; Munn et al.,Oncogene, 10:1653-1657, 1995], etc.). In addition, mutations in the 1pregion are thought to correlate with lymph node metastasis and tumorsize [Borg et al., Genes Chromosomes Cancer, 5:311-320, 1992; Tsukamotoet al., Cancer, 82:317-322, 1998]. Moreover, the genetic mutationassociated with endodermal sinus tumors (CESTs) developed in smallchildren under four years is proposed to occur on chromosome 1p [Perlmanet al., Genes Chromosomes Cancer, 16:15-20, 1996]. These facts indicatethat one or more genetic mutations in chromosome 1p are associated withmalignant tumors. However, the causative gene has not yet beendiscovered.

SUMMARY

The object of the present invention is to provide a novel proteininvolved in the proliferation mechanism of the cells and the geneencoding the protein, as well as methods for producing and using thesame.

The present inventors screened the mouse RS-4 cell cDNA libraryaccording to the immunoscreening method using antibodies against proteinp33, which is about 30 kDa in size and contained in the Triton X-100insoluble fraction of the immortalized cell (NIH3T3) plasma membraneP100 fraction. Using thus obtained cDNA as a probe, human testis librarywas screened and the inventors succeeded in cloning the novel gene,Gros1, from the library. Two types of human Gros1 cDNAs (SEQ ID NOs:1and 3) exist: one encodes a protein consisting of 363 amino acids(designated “human Gros1-S protein”, SEQ ID NO:2), and the other encodesa protein consisting of 736 amino acids (designated “human Gros1-Lprotein”, SEQ ID NO:4).

Moreover, using the cDNAs obtained by the above immunoscreening methodas a probe, mouse testis library was both screened and searched forESTs, to successfully identify mouse Gros1-L cDNA (SEQ ID NO:5) andmouse Gros1-S cDNA (SEQ ID NO:7). They encode a protein consisting of747 amino acids (SEQ ID NO:6) and 542 amino acids (SEQ ID NO:8),respectively.

Human and mouse Gros1s were found to be homologous to the novel basementmembrane-associated proteoglycan found in the data bank, leprecan,isolated from rat cDNA (Wassenhove-McCarthy et al., J. Biol. Chem.,274:25004-25017, 1999). In addition, as a result of the motif searchanalysis of amino acid sequences, the amino acid sequences of mouse andhuman Gros1-Ls were found to comprise the leucine zipper structure oftenobserved in some members of the transcription factors.

As the result of chromosome mapping of human Gros1, the Gros1 gene wasfound to exist on the human chromosome 1 short arm (1p), a sitesuggested to have non-random mutations in many malignant tumors (Caron,Med. Pediatr. Oncol., 24:215-221, 1995; Schwab et al., Genes ChromosomesCancer, 16:211-229, 1996).

The amount of Gros1 mRNA expressed in tissues, cells and those duringdevelopmental stages was detected by Northern blot analysis. As aresult, in human, 4.4 kb and 2.5 kb bands were strongly expressed intestis, ovary and placenta, and weakly in most other tissues (FIG. 4).In addition, mRNA expression was higher in cultured human cells than inabove tissues, and, in human normal cultured cells, the expression ofthe 2.5 kb mRNA was almost 10 times higher than that for the 4.4 kb mRNA(FIG. 5). In mouse, 3.5 kb and 2.5 kb bands were weakly expressed inmost tissues, not expressed in brain or spleen; only the 2.5 kb band wasexpressed in the testis. Accordingly, in the testis and ovary, only theshorter form among the two transcripts of the Gros1 genes was detected.The expression during the developmental process was shown todramatically disappear on the 11th day of the developmental process(FIG. 6).

The present inventors performed a function analysis of Gros1 byintroducing the gene encoding the mouse 85 kDa protein (Gros1-L; SEQ IDNO:6) into NIH3T3 cells. As a result, cell proliferation was repressedand colony forming activity was decreased in cells expressing thefull-length Gros1-L as compared to those of the control and Gros1 whoseC-terminus is deleted. On the other hand, in cells in which antisenseRNA of Gros1-L was expressed, the colony forming activity increased 5folds.

Based on these analyses, Gros1 proteins are thought to be novel genesinvolved in the control of cell proliferation, and related to thedevelopment and growth of tumors. Thus, Gros1 proteins are useful astools for developing pharmaceuticals against tumors.

The present invention relates to novel proteins (Gros1) involved in cellproliferation and the genes encoding them, as well as the production andthe use the same. More specifically, the present invention provides thefollowing:

1. A DNA of any one of the following (a) to (d):

-   -   (a) a DNA encoding the protein consisting of the amino acid        sequence of any one of SEQ ID NOs:2, 4, 6, or 8;    -   (b) a DNA containing the coding region of the nucleotide        sequence of any one of SEQ ID NOs:1, 3, 5, or 7;    -   (c) a DNA encoding a protein consisting of the amino acid        sequence of any one of SEQ ID NOs:2, 4, 6, or 8, in which one or        more amino acids are replaced, deleted, inserted, and/or added,        the encoded protein being functionally equivalent to the protein        consisting of the amino acid sequence of any one of SEQ ID        NOs:2, 4, 6, or 8; and    -   (d) a DNA hybridizing under stringent conditions with a DNA        consisting of the nucleotide sequence of any one of SEQ ID        NOs:1, 3, 5, or 7, and encoding a protein functionally        equivalent to the protein consisting of the amino acid sequence        of any one of SEQ ID NOs:2, 4, 6, or 8.

2. A DNA encoding a partial peptide of the protein consisting of theamino acid sequence of any one of SEQ ID NOs:2, 4, 6, or 8.

3. A vector into which the DNA of (1) or (2) is inserted.

4. A transformed cell harboring the DNA of (1) or (2), or the vector of(3).

5. A protein or peptide encoded by the DNA of (1) or (2).

6. A method for producing the protein or peptide of (5), comprising thesteps of culturing the transformed cell of (4), and collecting theprotein expressed from the cells or the culture supernatant thereof.

7. An antibody binding to the protein of (5).

8. A polynucleotide hybridizing with the DNA consisting of thenucleotide sequence of any one of SEQ ID NOs:1, 3, 5, or 7 or thecomplementary strand thereof, and comprising at least 15 nucleotides.

9. A method of screening for a compound that binds to the protein of(5), comprising the steps of:

-   -   (a) contacting a subject sample with the protein or the partial        peptide thereof;    -   (b) detecting the binding activity of the subject sample with        the protein or the partial peptide thereof; and    -   (c) selecting the test compound that binds to the protein or the        partial peptide thereof.

10. A compound binding to the protein of (5), which can be isolated bythe method of (9).

11. A method of screening for a compound that promotes or inhibits theactivity of the protein of (5), comprising the steps of:

-   -   (a) culturing cells which express the protein or the partial        peptide thereof in the presence of a subject sample;    -   (b) detecting the proliferation of the cell; and    -   (c) selecting the compound that promotes or inhibits the        proliferation as compared to the proliferation detected in the        absence of the subject sample.

12. A compound that promotes or inhibits the activity of the protein of(5), which can be isolated by the method of (11).

The present invention provides a novel protein Gros1 involved in thecell proliferation mechanism. SEQ ID NOs:1 and 3 show nucleotidesequences of the cDNA of two types of human Gros1 (human Gros1-S andhuman Gros1-L, respectively) isolated by the inventors, and SEQ ID NOs:2and 4 show the amino acids sequences encoded by the cDNAs, respectively.SEQ ID NOs:5 and 7 show nucleotide sequences of the cDNA of two types ofmouse Gros1 (mouse Gros1-L and mouse Gros1-S, respectively) isolated bythe present inventors, and SEQ ID NOs:6 and 8 show the amino acidssequences encoded by the cDNAs, respectively.

When the Gros1-L protein was expressed exogenously in NIH-3T3 cells,cell proliferation was inhibited and the colony forming activitydecreased. In contrast, when the expression of Gros1-L protein wasrepressed by the introduction of Gros1 antisense cDNA in NIH-3T3 cells,the colony forming activity increased dramatically. Thus, the Gros1protein is considered to be involved in the control of cellproliferation. This is also supported by the fact that human Gros1 geneis present on chromosome 1 short arm (1p), a site that is purportedlyassociated with malignant tumors. Therefore, the Gros1 proteins of thepresent invention can be conveniently used as tools, for purifying orcloning factors controlling cell proliferation, as well as targets, forexample, for screening candidate compounds of drugs useful for treatingor preventing disorders related to cell proliferation, such as tumors.Moreover, the Gros1 genes can be applied in the treatment, for example,gene therapy for various tumors.

The present invention encompasses proteins functionally equivalent tothe Gros1 proteins. Such proteins include, for example, homologousproteins of other organisms corresponding to the human or mouse Gros1protein, as well as mutants of human or mouse Gros1 proteins.

In the present invention, the term “functionally equivalent” means thatthe subject protein has the activity to inhibit cell proliferation likeGros1 proteins. Whether the subject protein has a cell proliferationinhibitory activity or not can be judged by introducing the DNA encodingthe subject protein into a cell, such as NIH-3T3, expressing theprotein, and detecting repression of proliferation of the cells orreduction in colony forming activity.

Methods for preparing proteins functionally equivalent to a givenprotein are well known by a person skilled in the art and include knownmethods of introducing mutations into the protein. For example, oneskilled in the art can prepare proteins functionally equivalent to thehuman or mouse Gros1 protein by introducing an appropriate mutation inthe amino acid sequence of the human or mouse Gros1 protein bysite-directed mutagenesis (Hashimoto-Gotoh et al., Gene, 152:271-275,1995; Zoller et al., Methods Enzymol., 100:468-500, 1983; Kramer et al.,Nucleic Acids Res., 12:9441-9456, 1984; Kramer et al., Methods Enzymol.,154:350-367, 1987; Kunkel, Proc. Natl. Acad. Sci. USA, 82:488-492, 1985;Kunkel, Methods Enzymol., 85:2763-2766, 1988). Amino acid mutations canoccur in nature, too. The protein of the present invention includesthose proteins having the amino acid sequences of the human or mouseGros1 protein in which one or more amino acids are mutated, provided theresulting mutated proteins are functionally equivalent to the human ormouse Gros1 protein. The number of amino acids to be mutated in such amutant is generally 10 amino acids or less, preferably 6 amino acids orless, and more preferably 3 amino acids or less.

Mutated or modified proteins, proteins having amino acid sequencesmodified by deleting, adding and/or replacing one or more amino acidresidues of a certain amino acid sequence, have been known to retain theoriginal biological activity (Mark et al., Proc. Natl. Acad. Sci. USA,81:5662-5666, 1984; Zoller et al., Nucleic Acids Res., 10:6487-6500,1982; Wang et al., Science, 224:1431-1433; Dalbadie-McFarland et al.,Proc. Natl. Acad. Sci. USA, 79:6409-6413, 1982).

The amino acid residue to be mutated is preferably mutated into adifferent amino acid in which the properties of the amino acidside-chain are conserved. Examples of properties of amino acid sidechains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and sidechains having the following functional groups or characteristics incommon: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl groupcontaining side-chain (S, T, Y); a sulfur atom containing side-chain (C,M); a carboxylic acid and amide containing side-chain (D, N, E, Q); abase containing side-chain (R, K, H); and an aromatic containingside-chain (H, F, Y, W). (The parenthetic letters indicate theone-letter codes of amino acids).

An example of a protein to which one or more amino acids residues areadded to the amino acid sequence of human or mouse Gros1 protein (SEQ IDNOs:2, 4, 6, or 8) is a fusion protein containing the human or mouseGros1 protein. Fusion proteins are, fusions of the human or mouse Gros1protein and other peptides or proteins, and are included in the presentinvention. Fusion proteins can be made by techniques well known to aperson skilled in the art, such as by linking the DNA encoding the humanor mouse Gros1 protein of the invention with DNA encoding other peptidesor proteins, so that the frames match, inserting the fusion DNA into anexpression vector and expressing it in a host. There is no restrictionas to the peptides or proteins fused to the protein of the presentinvention. Known peptides that can be used as peptides that are fused tothe protein of the present invention include, for example, FLAG (Hopp etal., Biotechnology, 6:1204-1210, 1988), 6×His containing six His(histidine) residues, 10×His, HA (Influenza agglutinin), human c-mycfragment, VSP-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag,SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, Protein Cfragment, and the like. Examples of proteins that may be fused to aprotein of the invention include GST (glutathione-S-transferase), HA(Influenza agglutinin), immunoglobulin constant region, β-galactosidase,MBP (maltose-binding protein), and such.

Fusion proteins can be prepared by fusing commercially available DNA,encoding the fusion peptides or proteins discussed above, with the DNAencoding the protein of the present invention and expressing the fusedDNA prepared.

An alternative method known in the art to isolate functionallyequivalent proteins is, for example, the method using a hybridizationtechnique (Sambrook et al., Molecular Cloning 2nd ed. 9.47-9.58, ColdSpring Harbor Lab. Press, 1989). One skilled in the art can readilyisolate a DNA having high homology with a whole or part of the DNAsequence (SEQ ID NOs:1, 3, 5 or 7) encoding the human or mouse Gros1protein, and isolate functionally equivalent proteins to the human ormouse Gros1 protein from the isolated DNA. The proteins of the presentinvention include those that are encoded by DNA that hybridize with awhole or part of the DNA sequence encoding the human or mouse Gros1protein and are functionally equivalent to the human or mouse Gros1protein. These proteins include mammal homologues corresponding to theprotein derived from human or mouse (for example, a protein encoded by amonkey, rat, rabbit and bovine gene). In isolating a cDNA highlyhomologous to the DNA encoding the human or mouse Gros1 protein fromanimals, it is particularly preferable to use tissues from ovary ortestis.

The condition of hybridization for isolating a DNA encoding a proteinfunctionally equivalent to the human or mouse Gros1 protein can beroutinely selected by a person skilled in the art. For example,hybridization may be performed by conducting prehybridization at 68° C.for 30 min or longer using “Rapid-hyb buffer” (Amersham LIFE SCIENCE),adding a labeled probe, and warming at 68° C. for 1 hour or longer. Thefollowing washing step can be conducted, for example, in a low stringentcondition. A low stringent condition is, for example, 42° C., 2×SSC,0.1% SDS, or preferably 50° C., 2×SSC, 0.1% SDS. More preferably, highstringent conditions are used. A high stringent condition is, forexample, washing 3 times in 2×SSC, 0.01% SDS at room temperature for 20min, then washing 3 times in 1×SSC, 0.1% SDS at 37° C. for 20 min, andwashing twice in 1×SSC, 0.1% SDS at 50° C. for 20 min. However, severalfactors such as temperature and salt concentration can influence thestringency of hybridization and one skilled in the art can suitablyselect the factors to achieve the requisite stringency.

In place of hybridization, a gene amplification method, for example, thepolymerase chain reaction (PCR) method, can be utilized to isolate a DNAencoding a protein functionally equivalent to the human or mouse Gros1protein, using a primer synthesized based on the sequence information ofthe DNA (SEQ ID NO:1, 3, 5 or 7) encoding the human or mouse Gros1protein.

Proteins that are functionally equivalent to the human or mouse Gros1protein encoded by the DNA isolated through the above hybridizationtechniques or gene amplification techniques, normally have a highhomology to the amino acid sequence of the human or mouse Gros1 protein.“High homology” typically refers to a homology of 40% or higher,preferably 60% or higher, more preferably 80% or higher, even morepreferably 95% or higher. The homology of a protein can be determined byfollowing the algorithm in “Wilbur, W. J. and Lipman, D. J. Proc. Natl.Acad. Sci. USA (1983) 80, 726-730”.

A protein of the present invention may have variations in amino acidsequence, molecular weight, isoelectric point, the presence or absenceof sugar chains, or form, depending on the cell or host used to produceit or the purification method utilized.

Nevertheless, so long as it has a function equivalent to that of thehuman or mouse Gros1 protein (SEQ ID NO:2, 4, 6 or 8) of the presentinvention, it is within the scope of the present invention.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. For example, the substantially purepolypeptide is at least 75%, 80, 85, 95, or 99% pure by dry weight.Purity can be measured by any appropriate standard method known in theart, for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

Accordingly, the invention includes a polypeptide having a sequenceshown as SEQ ID NO:2, 4, 6 or 8. The invention also includes apolypeptide, or fragment thereof, that differs from the correspondingsequence shown as SEQ ID NO:2, 4, 6 or 8. The differences are,preferably, differences or changes at a non-essential residue or aconservative substitution. In one embodiment, the polypeptide includesan amino acid sequence at least about 60% identical to a sequence shownas SEQ ID NO:2, 4, 6 or 8, or a fragment thereof. Preferably, thepolypeptide is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% ormore identical to SEQ ID NO:2, 4, 6 or 8 and has at least one cellproliferation related function or activity described herein, e.g., thepolypeptide inhibits cell proliferation or decreases colony formingactivity. Preferred polypeptide fragments of the invention are at least10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, or more, of thelength of the sequence shown as SEQ ID NO:2, 4, 6 or 8 and have at leastone cell proliferation related function or activity described herein. Oralternatively, the fragment can be merely an immunogenic fragment.

The proteins of the present invention can be prepared as recombinantproteins or natural proteins, by methods well known to those skilled inthe art. A recombinant protein can be prepared by inserting a DNA, whichencodes the protein of the present invention (for example, the DNAcomprising the nucleotide sequence of SEQ ID NOs:1, 3, 5, or 7), into anappropriate expression vector, introducing the vector into anappropriate host cell, collecting thus obtained recombinants, obtainingthe extract thereof, and purifying the protein by subjecting the extractto chromatography, for example, ion exchange chromatography, reversephase chromatography, gel filtration, or affinity chromatographyutilizing a column to which antibodies against the protein of thepresent invention is fixed, or by combining more than one ofaforementioned columns.

Also when the protein of the present invention is expressed within hostcells (for example, animal cells and E. coli) as a fusion protein withglutathione-S-transferase protein or as a recombinant proteinsupplemented with multiple histidines, the expressed recombinant proteincan be purified using a glutathione column or nickel column.

After purifying the fusion protein, it is also possible to excluderegions other than the objective protein by cutting with thrombin orfactor-Xa as required.

A natural protein can be isolated by methods known to a person skilledin the art, for example, by contacting the affinity column, in whichantibodies binding to the Gros1 protein described below are bound, withthe extract of tissues or cells expressing the protein of the presentinvention. The antibodies can be polyclonal antibodies or a monoclonalantibodies.

The present invention also encompasses partial peptides of the proteinof the present invention. The partial peptide has an amino acid sequencespecific to the protein of the present invention and consists of atleast 7 amino acids, preferably 8 amino acids or more, and morepreferably 9 amino acids or more. The partial peptide can be used, forexample, for preparing antibodies against the protein of the presentinvention, screening for a compound that binds to the protein of thepresent invention, and screening for accelerators or inhibitors of theprotein of the present invention.

A partial peptide of the invention can be produced by geneticengineering, by known methods of peptide synthesis, or by digesting theprotein of the invention with an appropriate peptidase. For peptidesynthesis, for example, solid phase synthesis or liquid phase synthesismay be used.

Furthermore, the present invention provides DNA encoding the proteins ofthe present invention. The DNA of the present invention can be used forthe in vivo or in vitro production of the protein of the presentinvention as described above, or can be applied to gene therapy fordiseases attributed to genetic abnormality in the gene encoding theprotein of the present invention. Any form of the DNA of the presentinvention can be used, so long as it encodes the protein of the presentinvention. Specifically, cDNA synthesized from the mRNA, genomic DNA,and chemically synthesized DNA can be used. The DNA of the presentinvention include a DNA comprising a given nucleotide sequences as wellas its degenerate sequences, so long as the resulting DNA encodes aprotein of the present invention.

The DNA of the present invention can be prepared by methods known to aperson skilled in the art. For example, the DNA of the present inventioncan be prepared by: preparing a cDNA library from cells which expressthe protein of the present invention, and conducting hybridization usinga partial sequence of the DNA of the present invention (for example, SEQID NOs:1, 3, 5, or 7) as a probe. A cDNA library can be prepared, forexample, by the method described in Sambrook et al., Molecular Cloning,Cold Spring Harbor Laboratory Press (1989); alternatively, commerciallyavailable cDNA libraries may be used. A cDNA library can be alsoprepared by: extracting RNAs from cells expressing the protein of thepresent invention, synthesizing oligo DNAs based on the sequence of theDNA of the present invention (for example, SEQ ID NOs:1, 3, 5 or 7),conducting PCR by using the oligos as primers, and amplifying cDNAsencoding the protein of the present invention.

As used herein, an “isolated nucleic acid” is a nucleic acid, thestructure of which is not identical to that of any naturally occurringnucleic acid or to that of any fragment of a naturally occurring genomicnucleic acid spanning more than three genes. The term therefore covers,for example, (a) a DNA which has the sequence of part of a naturallyoccurring genomic DNA molecule but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Specifically excluded from this definition are nucleicacids present in random, uncharacterized mixtures of different DNAmolecules, transfected cells, or cell clones, e.g., as these occur in aDNA library such as a cDNA or genomic DNA library.

Accordingly, in one aspect, the invention provides an isolated orpurified nucleic acid molecule that encodes a polypeptide describedherein or a fragment thereof. Preferably, the isolated nucleic acidmolecule includes a nucleotide sequence that is at least 60% identicalto the nucleotide sequence shown in SEQ ID NO:1, 3, 5 or 7. Morepreferably, the isolated nucleic acid molecule is at least 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, identical to the nucleotide sequence shown in SEQ ID NO:1, 3, 5 or7. In the case of an isolated nucleic acid molecule which is longer thanor equivalent in length to the reference sequence, e.g., SEQ ID NO:1, 3,5 or 7, the comparison is made with the full length of the referencesequence. Where the isolated nucleic acid molecule is shorter that thereference sequence, e.g., shorter than SEQ ID NO:1, 3, 5 or 7, thecomparison is made to a segment of the reference sequence of the samelength (excluding any loop required by the homology calculation).

As used herein, “% identity” of two amino acid sequences, or of twonucleic acid sequences, is determined using the algorithm of Karlin andAltschul (Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990), modified asin Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877,1993)). Such an algorithm is incorporated into the NBLAST and XBLASTprograms of Altschul et al. (J. Mol. Biol., 215:403-410, 1990). BLASTnucleotide searches are performed with the NBLAST program, score=100,wordlength=12. BLAST protein searches are performed with the XBLASTprogram, score=50, wordlength=3. To obtain gapped alignment forcomparison purposes GappedBLAST is utilized as described in Altschul etal. (Nucleic Acids Res., 25:3389-3402, 1997). When utilizing BLAST andGappedBLAST programs the default parameters of the respective programs(e.g., XBLAST and NBLAST) are used to obtain nucleotide sequenceshomologous to a nucleic acid molecule of the invention.

In addition, by sequencing the nucleotides of the obtained cDNA, thetranslation region encoded by the cDNA can be determined, and the aminoacid sequence of the protein of the present invention can be obtained.Moreover, by screening the genomic DNA library using the obtained cDNAas a probe, the genomic DNA can be isolated.

More specifically, mRNAs may first be prepared from cells, tissue, ororgan (for example, ovary, testis, placenta, etc.) in which the proteinof the invention is expressed. Known methods can be used to isolatemRNAs; for instance, total RNA may be prepared by guanidineultracentrifugation (Chirgwin et al., Biochemistry, 18:5294-5299, 1979)or AGPC method (Chomczynski et al., Anal. Biochem., 162:156-159, 1987).In addition, mRNA may be purified from total RNA using mRNA PurificationKit (Pharmacia) and such or, alternatively, mRNA may be directlypurified by QuickPrep mRNA Purification Kit (Pharmacia).

The obtained mRNA is used to synthesize cDNA using reversetranscriptase. cDNA may be synthesized by using a commercially availablekit, such as the AMV Reverse Transcriptase First-strand cDNA SynthesisKit (Seikagaku Kogyo). Alternatively, cDNA may be synthesized andamplified following the 5′-RACE method (Frohman et al., Proc. Natl.Acad. Sci. USA, 85:8998-9002, 1988; Belyaysky et al., Nucleic AcidsRes., 17:2919-2932, 1989), which uses a primer and such, describedherein, the 5′-Ampli FINDER RACE Kit (Clontech), and polymerase chainreaction (PCR).

A desired DNA fragment is prepared from the PCR products and ligatedwith a vector DNA. The recombinant vectors are used to transform E. coliand such, and a desired recombinant vector is prepared from a selectedcolony. The nucleotide sequence of the desired DNA can be verified byconventional methods, such as dideoxynucleotide chain termination.

The nucleotide sequence of a DNA of the invention may be designed to beexpressed more efficiently by taking into account the frequency of codonusage in the host to be used for expression (Grantham et al., NucleicAcids Res., 9:43-74, 1981). The DNA of the present invention may bealtered by a commercially available kit or a conventional method. Forinstance, the DNA may be altered by digestion with restriction enzymes,insertion of a synthetic oligonucleotide or an appropriate DNA fragment,addition of a linker, or insertion of the initiation codon (ATG) and/orthe stop codon (TAA, TGA, or TAG).

Specifically, the DNA of the present invention encompasses the DNAcomprising the nucleotide sequence from base A at position 52 to base Cat position 1140 of SEQ ID NO: 1; the DNA from base A at position 52 tobase A at position 2259 of SEQ ID NO:3; the DNA from base A at position12 to base G at position 2252 of SEQ ID NO:5; and that from base A atposition 12 to base A at position 1640 of SEQ ID NO:3.

Furthermore, the present invention provides a DNA that hybridizes understringent conditions with a DNA having a nucleotide sequence of SEQ IDNOs:1, 3, 5 or 7, and encodes a protein functionally equivalent to theprotein of the invention described above.

One skilled in the art may appropriately choose stringent conditions.For example, low stringent condition can be used. More preferably, highstringent condition can be used. These conditions are the same as thosedescribed above. The hybridizing DNA above is preferably a cDNA or achromosomal DNA.

The present invention also provides a vector into which a DNA of thepresent invention is inserted. A vector of the present invention isuseful to keep a DNA of the present invention in host cell, or toexpress the protein of the present invention. When E. coli is a hostcell and the vector is amplified and produced in a large amount in E.coli (e.g., JM109, DH5α, HB101, or XL1Blue), the vector should have“ori” to be amplified in E. coli and a marker gene for selectingtransformed E. coli (e.g., a drug-resistance gene selected by a drugsuch as ampicillin, tetracycline, kanamycin, chloramphenicol or thelike). For example, M13-series vectors, pUC-series vectors, pBR322,pBluescript, pCR-Script, etc., can be used. In addition, pGEM-T,pDIRECT, and pT7 can also be used for subcloning and extracting cDNA aswell as the vectors described above. When a vector is used to producethe protein of the present invention, an expression vector is especiallyuseful. For example, an expression vector to be expressed in E. colishould have the above characteristics to be amplified in E. coli. WhenE. coli, such as JM109, DH5α, HB101, or XL1 Blue, are used as a hostcell, the vector should have a promoter, for example, lacZ promoter(Ward et al., Nature, 341:544-546, 1989; FASEB J, 6:2422-2427, 1992),araB promoter (Better et al., Science, 240:1041-1043, 1988), or T7promoter or the like, that can efficiently express the desired gene inE. coli. In that respect, pGEX-5X-1 (Pharmacia), “QIAexpress system”(Qiagen), pEGFP and pET (in this case, the host is preferably BL21 whichexpresses T7 RNA polymerase), for example, can be used instead of theabove vectors.

Additionally, the vector may also contain a signal sequence forpolypeptide secretion. An exemplary signal sequence that directs theprotein to be secreted to the periplasm of the E. coli is the pelBsignal sequence (Lei et al., J. Bacteriol., 169: 4379, 1987). Means forintroducing the vectors into the target host cells include, for example,the calcium chloride method, and the electroporation method.

In addition to E. coli, for example, expression vectors derived frommammals (for example, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids.Res., 18(17):5322, 1990), pEF, pCDM8), expression vectors derived frominsect cells (for example, “Bac-to-BAC baculovirus expression system”(GIBCO BRL), pBacPAK8), expression vectors derived from plants (forexample pMH1, pMH2), expression vectors derived from animal viruses (forexample, pHSV, pMV, pAdexLcw), expression vectors derived fromretroviruses (for example, pZIpneo), expression vector derived fromyeast (for example, “Pichia Expression Kit” (Invitrogen), pNV11,SP-Q01), and expression vectors derived from Bacillus subtilis (forexample, pPL608, pKTH50) can be used for producing the protein of thepresent invention.

In order to express the vector in animal cells, such as CHO, COS, orNIH3T3 cells, the vector should have a promoter necessary for expressionin such cells, for example, the SV40 promoter (Mulligan et al., Nature(1979) 277, 108), the MMLV-LTR promoter, the EF1a promoter (Mizushima etal., Nucleic Acids Res. (1990) 18, 5322), the CMV promoter, and thelike, and preferably a marker gene for selecting transformants (forexample, a drug resistance gene selected by a drug (e.g., neomycin,G418)). Examples of known vectors with these characteristics include,for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

In addition, methods may be used to express a gene stably and, at thesame time, to amplify the copy number of the gene in cells. For example,a vector comprising the complementary DHFR gene (for example pCHO I) maybe introduced into CHO cells in which the nucleic acid synthesizingpathway is deleted, and then amplified by methotrexate (MTX).Furthermore, in case of transient expression of a gene, the methodwherein a vector comprising a replication origin of SV40 (pcD, etc.) istransfected into COS cells comprising the SV40 T antigen expressing geneon the chromosome can be used.

The DNA of the present invention can further be expressed in vivo inanimals, for example, by inserting the DNA of the present invention intoan appropriate vector and introducing it into living bodies by methodssuch as the retrovirus method, the liposome method, the cationicliposome method, and the adenovirus method. In such a manner, genetherapy against diseases attributed to mutation of Gros1 gene of thepresent invention can be accomplished. As a vector to be used, forexample, adenovirus vector (for example pAdexlcw), and retrovirus vector(for example, pZIPneo) can be mentioned, but is not restricted thereto.General gene manipulation, such as insertion of the DNA of the presentinvention to a vector, can be performed according to conventionalmethods (Molecular Cloning, 5:61-65. 63). Administration into a livingbody can be either an ex vivo method, or in vivo method.

The present invention further provides a host cell into which the vectorof the present invention has been transfected. The host cell into whichthe vector of the invention is transfected is not particularly limited.For example, E. coli, various animal cells and such can be used. Thehost cells of the present invention can be used, for example, as aproduction system for producing or expressing the protein of the presentinvention. The present invention provides methods of producing a proteinof the invention both in vitro and in vivo. For in vitro production,eukaryotic cells or prokaryotic cells can be used as host cells.

Useful eukaryotic cells may be animal, plant, or fungi cells. Exemplaryanimal cells include, for example, mammalian cells such as CHO (J. Exp.Med., 108:945, 1995), COS, 3T3, myeloma, baby hamster kidney (BHK),HeLa, or Vero cells, amphibian cells such as Xenopus oocytes (Valle etal., Nature, 291:340-358, 1981), or insect cells such as sf9, sf21, orTn5 cells. CHO cells lacking DHFR gene (dhfr-CHO) (Proc. Natl. Acad.Sci. USA, 77:4216-4220, 1980) or CHO K-1 (Proc. Natl. Acad. Sci. USA,60:1275, 1968) may also be used. Of the animal cells, CHO cells areparticularly preferable for the mass expression. A vector can betransfected into host cells by, for example, the calcium phosphatemethod, the DEAE-dextran method, the cationic liposome DOTAP (BoehringerMannheim), the electroporation method, the lipofection method, and soon.

As plant cells, plant cells originating from Nicotiana tabacum are knownas protein-production systems, and may be used as callus cultures. Asfungi cells, yeast cells such as Saccharomyces, including Saccharomycescerevisiae, or filamentous fungi such as Aspergillus, includingAspergillus niger, are known and may be used herein.

Useful prokaryotic cells include bacterial cells, such as E. coli, forexample, JM109, DH5α, and HB101. Other bacterial systems include,Bacillus subtilis.

These cells are transformed by a desired DNA, and the resultingtransformants are cultured in vitro to obtain the protein. Transformantscan be cultured using known methods. Culture medium for animal cellsinclude, for example, DMEM, MEM, RPMI1640, or IMDM may be used with orwithout serum supplement such as fetal calf serum (FCS). The pH of theculture medium is preferably between about 6 and 8. Such cells aretypically cultured at about 30 to 40° C. for about 15 to 200 hr, and theculture medium may be replaced, aerated, or stirred if necessary.

Animal and plant hosts may be used for in vivo production. For example,a desired DNA can be transfected into an animal or plant host. Encodedproteins are produced in vivo, and then recovered. These animal andplant hosts are included in host cells of the present invention.

Animals to be used for the production system described above include,but are not limited to, mammals and insects. Mammals, such as goat,porcine, sheep, mouse, and bovine, may be used (Vicki Glaser, SPECTRUMBiotechnology Applications (1993)). Alternatively, the mammals may betransgenic animals.

For instance, a desired DNA may be prepared as a fusion gene, by fusingit with a gene, such as goat β casein gene which encodes a proteinspecifically produced into milk. DNA fragments comprising the fusiongene are injected into goat embryos, which are then impregnated intofemale goats. Proteins are recovered from milk produced by thetransgenic goats (i.e., those born from the goats that had received themodified embryos) or from their offspring. To increase the amount ofmilk containing the proteins produced by transgenic goats, appropriatehormones may be administered to them (Ebert et al., Bio/Technology,12:699-702, 1994).

Alternatively, insects, such as the silkworm, may be used. A DNAencoding a desired protein inserted into baculovirus can be used totransfect silkworms, and the desired protein may be recovered from theirbody fluid (Susumu et al., Nature, 315:592-594, 1985).

As plants, for example, tobacco can be used. In use of tobacco, a DNAencoding a desired protein may be inserted into a plant expressionvector, such as pMON530, which is introduced into bacteria, such asAgrobacterium tumefaciens. Then, the bacteria is used to transfect atobacco plant, such as Nicotiana tabacum, and a desired polypeptide isrecovered from their leaves (Julian et al., Eur. J. Immunol.,24:131-138, 1994).

A protein of the present invention obtained as above may be isolatedfrom inside or outside (such as medium) of host cells, and purified as asubstantially pure homogeneous protein. The method for protein isolationand purification is not limited to any specific method; in fact, anystandard method may be used. For instance, column chromatography,filter, ultrafiltration, salt precipitation, solvent precipitation,solvent extraction, distillation, immunoprecipitation,SDS-polyacrylamide gel electrophoresis, isoelectric pointelectrophoresis, dialysis, and recrystallization may be appropriatelyselected and combined to isolate and purify the protein.

Examples of chromatography include, for example, affinitychromatography, ion-exchange chromatography, hydrophobic chromatography,gel filtration, reverse phase chromatography, adsorption chromatography,and such (Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed. Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press (1996)). These chromatographies may be performedby liquid chromatography, such as HPLC and FPLC. Thus, the presentinvention provides for highly purified proteins prepared by the abovemethods.

A protein of the present invention may be optionally modified orpartially deleted by treating it with an appropriate proteinmodification enzyme before or after purification. Useful proteinmodification enzymes include, but are not limited to, trypsin,chymotrypsin, lysylendopeptidase, protein kinase, glucosidase and so on.

The present invention provides an antibody that binds to the protein ofthe invention. The antibody of the invention can be used in any form,such as monoclonal or polyclonal antibodies, and includes antiserumobtained by immunizing an animal such as a rabbit with the protein ofthe invention, all classes of polyclonal and monoclonal antibodies,human antibodies, and humanized antibodies produced by geneticrecombination.

A protein of the invention used as an antigen to obtain an antibody maybe derived from any animal species, but preferably is derived from amammal such as a human, mouse, or rat, more preferably from a human. Ahuman-derived protein may be obtained from the nucleotide or amino acidsequences disclosed herein.

According to the present invention, the protein to be used as animmunization antigen may be a complete protein or a partial peptide ofthe protein. A partial peptide may comprise, for example, the amino(N)-terminal or carboxy (C)-terminal fragment of a protein of thepresent invention. Herein, an antibody is defined as a protein thatreacts with either the full length or a fragment of a protein of thepresent invention.

A gene encoding a protein of the invention or its fragment may beinserted into a known expression vector, which is then used to transforma host cell as described herein. The desired protein or its fragment maybe recovered from the outside or inside of host cells by any standardmethod, and may subsequently be used as an antigen. Alternatively, cellsexpressing the protein or their lysates, or a chemically synthesizedprotein may be used as the antigen.

Any mammalian animal may be immunized with the antigen, but preferablythe compatibility with parental cells used for cell fusion is taken intoaccount. In general, animals of Rodentia, Lagomorpha, or Primates areused.

Animals of Rodentia include, for example, mouse, rat, and hamster.Animals of Lagomorpha include, for example, rabbit. Animals of Primatesinclude, for example, a monkey of Catarrhini (old world monkey) such asMacaca fascicularis, rhesus monkey, sacred baboon, and chimpanzees.

Methods for immunizing animals with antigens are known in the art.Intraperitoneal injection or subcutaneous injection of antigens is astandard method for immunization of mammals. More specifically, antigensmay be diluted and suspended in an appropriate amount of phosphatebuffered saline (PBS), physiological saline, etc. If desired, theantigen suspension may be mixed with an appropriate amount of a standardadjuvant, such as Freund's complete adjuvant, made into emulsion, andthen administered to mammalian animals. Preferably, it is followed byseveral administrations of antigen mixed with an appropriately amount ofFreund's incomplete adjuvant every 4 to 21 days. An appropriate carriermay also be used for immunization. After immunization as above, serum isexamined by a standard method for an increase in the amount of desiredantibodies.

Polyclonal antibodies against the proteins of the present invention maybe prepared by collecting blood from the immunized mammal examined forthe increase of desired antibodies in the serum, and by separating serumfrom the blood by any conventional method. Polyclonal antibodies includeserum containing the polyclonal antibodies, as well as the fractioncontaining the polyclonal antibodies may be isolated from the serum.Immunoglobulin G or M can be prepared from a fraction which recognizesonly the protein of the present invention using, for example, anaffinity column coupled with the protein of the present invention, andfurther purifying this fraction by using protein A or protein G column.

To prepare monoclonal antibodies, immune cells are collected from themammal immunized with the antigen and checked for the increased level ofdesired antibodies in the serum as described above, and are subjected tocell fusion. The immune cells used for cell fusion are preferablyobtained from spleen. Other preferred parental cells to be fused withthe above immunocyte include, for example, myeloma cells of mammalians,and more preferably myeloma cells having an acquired property for theselection of fused cells by drugs.

The above immunocyte and myeloma cells can be fused according to knownmethods, for example, the method of Milstein et al. (Galfre et al.,Methods Enzymol., 73:3-46, 1981).

Resulting hybridomas obtained by the cell fusion may be selected bycultivating them in a standard selection medium, such as HAT medium(hypoxanthine, aminopterin, and thymidine containing medium). The cellculture is typically continued in the HAT medium for several days toseveral weeks, the time being sufficient to allow all the other cells,with the exception of the desired hybridoma (non-fused cells), to die.Then, the standard limiting dilution is performed to screen and clone ahybridoma cell producing the desired antibody.

In addition to the above method in which a non-human animal is immunizedwith an antigen for preparing hybridoma, a hybridoma producing a desiredhuman antibody that is able to bind to the protein can be obtained bythe following method. First, human lymphocytes such as those infected byEB virus may be immunized with a protein, protein expressing cells, ortheir lysates in vitro. Then, the immunized lymphocytes are fused withhuman-derived myeloma cells that are capable of indefinitely dividing,such as U266, to yield the desired hybridoma (Unexamined PublishedJapanese Patent Application No. (JP-A) Sho 63-17688).

The obtained hybridomas are subsequently transplanted into the abdominalcavity of a mouse and the ascites are harvested. The obtained monoclonalantibodies can be purified by, for example, ammonium sulfateprecipitation, a protein A or protein G column, DEAE ion exchangechromatography, or an affinity column to which the protein of thepresent invention is coupled. The antibody of the present invention canbe used not only for purification and detection of the protein of thepresent invention, but also as a candidate for agonists and antagonistsof the protein of the present invention. In addition, this antibody canbe applied to the antibody treatment for diseases related to the proteinof the present invention. When the obtained antibody is to beadministered to the human body (antibody treatment), a human antibody ora humanized antibody is preferable for reducing immunogenicity.

For example, transgenic animals having a repertory of human antibodygenes may be immunized with an antigen selected from a protein, proteinexpressing cells, or their lysates. Antibody producing cells are thencollected from the animals and fused with myeloma cells to obtainhybridoma, from which human antibodies against the protein can beprepared (see WO92-03918, WO93-2227, WO94-02602, WO94-25585, WO96-33735,and WO96-34096).

Alternatively, an immune cell, such as an immunized lymphocyte,producing antibodies may be immortalized by an oncogene and used forpreparing monoclonal antibodies.

Monoclonal antibodies thus obtained can be also recombinantly preparedusing genetic engineering techniques (see, for example, Borrebaeck C. A.K. and Larrick J. W. Therapeutic Monoclonal Antibodies, published in theUnited Kingdom by MacMillan Publishers LTD (1990)). A DNA encoding anantibody may be cloned from an immune cell, such as a hybridoma or animmunized lymphocyte producing the antibody, inserted into anappropriate vector, and introduced into host cells to prepare arecombinant antibody. The present invention also provides recombinantantibodies prepared as described above.

Furthermore, an antibody of the present invention may be a fragment ofan antibody or modified antibody, so long as it binds to one or more ofthe proteins of the invention. For instance, the antibody fragment maybe Fab, F(ab′)₂, Fv, or single chain Fv (scFv), in which Fv fragmentsfrom H and L chains are ligated by an appropriate linker (Huston et al.,Proc. Natl. Acad. Sci. USA, 85:5879-5883, 1988). More specifically, anantibody fragment may be generated by treating an antibody with anenzyme, such as papain or pepsin. Alternatively, a gene encoding theantibody fragment may be constructed, inserted into an expressionvector, and expressed in an appropriate host cell (see, for example, Coet al., J. Immunol., 152:2968-2976, 1994; Better et al., MethodsEnzymol., 178:476-496, 1989; Pluckthun et al., Methods Enzymol.,178:497-515, 1989; Lamoyi, Methods Enzymol., 121:652-663, 1986;Rousseaux et al., Methods Enzymol., 121:663-669, 1986; Bird et al.,Trends Biotechnol., 9:132-137, 1991).

An antibody may be modified by conjugation with a variety of molecules,such as polyethylene glycol (PEG). The present invention provides forsuch modified antibodies. The modified antibody can be obtained bychemically modifying an antibody. These modification methods areconventional in the field.

Alternatively, an antibody of the present invention may be obtained as achimeric antibody, between a variable region derived from nonhumanantibody and the constant region derived from human antibody, or as ahumanized antibody, comprising the complementarity determining region(CDR) derived from nonhuman antibody, the frame work region (FR) derivedfrom human antibody, and the constant region. Such antibodies can beprepared by using known technology.

Antibodies obtained as above may be purified to homogeneity. Forexample, the separation and purification of the antibody can beperformed according to separation and purification methods used forgeneral proteins. For example, the antibody may be separated andisolated by the appropriately selected and combined use of columnchromatographies, such as affinity chromatography, filter,ultrafiltration, salting-out, dialysis, SDS polyacrylamide gelelectrophoresis, isoelectric focusing, and others (Antibodies: ALaboratory Manual. Ed Harlow and David Lane, Cold Spring HarborLaboratory, 1988), but are not limited thereto.

A protein A column and protein G column can be used as the affinitycolumn. Exemplary protein A columns to be used include, for example,Hyper D, POROS, and Sepharose F.F. (Pharmacia).

Exemplary chromatography, with the exception of affinity includes, forexample, ion-exchange chromatography, hydrophobic chromatography, gelfiltration, reverse-phase chromatography, adsorption chromatography, andthe like (Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press, 1996). The chromatographic procedures can becarried out by liquid-phase chromatography, such as HPLC, FPLC.

For example, measurement of absorbance, enzyme-linked immunosorbentassay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and/orimmunofluorescence may be used to measure the antigen binding activityof the antibody of the invention. In ELISA, the antibody of the presentinvention is immobilized on a plate, protein of the invention is appliedto the plate, and then a sample containing a desired antibody, such asculture supernatant of antibody producing cells or purified antibodies,is applied. Then, a secondary antibody that recognizes the primaryantibody and is labeled with an enzyme, such as alkaline phosphatase, isapplied, and the plate is incubated. Next, after washing, an enzymesubstrate, such as p-nitrophenyl phosphate, is added to the plate, andthe absorbance is measured to evaluate the antigen binding activity ofthe sample. A fragment of the protein, such as a C-terminal orN-terminal fragment may be used as a protein. BIAcore (Pharmacia) may beused to evaluate the activity of the antibody according to the presentinvention.

The above methods allow for the detection or measurement of the proteinof the invention, by exposing the antibody of the invention to a sampleassumed to contain the protein of the invention, and detecting ormeasuring the immune complex formed by the antibody and the protein.

Because the method of detection or measurement of the protein accordingto the invention can specifically detect or measure a protein, themethod may be useful in a variety of experiments in which the protein isused.

The present invention also provides a polynucleotide which hybridizeswith the DNA encoding human or mouse Gros1 protein (SEQ ID NOs:1, 3, 5,or 7) or the complementary strand thereof, and which comprises at least15 nucleotides. The polynucleotide of the present invention ispreferably a polynucleotide which specifically hybridizes with the DNAencoding the protein of the present invention. The term “specificallyhybridize” as used herein, means that cross-hybridization does not occursignificantly with DNA encoding other proteins, under the usualhybridizing conditions, preferably under stringent hybridizingconditions. Such polynucleotides include, probes, primers, nucleotidesand nucleotide derivatives (for example, antisense oligonucleotides andribozymes), which specifically hybridize with DNA encoding the proteinof the invention or its complementary strand. Moreover, suchpolynucleotide can be utilized for the preparation of DNA chip.

The present invention includes an antisense oligonucleotide thathybridizes with any site within the nucleotide sequence any one of SEQID NO:1, 3, 5 or 7. This antisense oligonucleotide is preferably againstat least 15 continuous nucleotides of the nucleotide sequence any one ofSEQ ID NO:1, 3, 5 or 7. The above-mentioned antisense oligonucleotide,which contains an initiation codon in the above-mentioned at least 15continuous nucleotides, is even more preferred.

Derivatives or modified products of antisense oligonucleotides can beused as antisense oligonucleotides. Examples of such modified productsinclude lower alkyl phosphonate modifications such asmethyl-phosphonate-type or ethyl-phosphonate-type, phosphorothioatemodifications and phosphoroamidate modifications.

The term “antisense oligonucleotides” as used herein means, not onlythose in which the nucleotides corresponding to those constituting aspecified region of a DNA or mRNA are entirely complementary, but alsothose having a mismatch of one or more nucleotides, as long as the DNAor mRNA and the antisense oligonucleotide can specifically hybridizewith the nucleotide sequence of SEQ ID NO:1, 3, 5 or 7.

Such polynucleotides are contained as those having, in the “at least 15continuous nucleotide sequence region”, a homology of at least 70% orhigher, preferably at 80% or higher, more preferably 90% or higher, evenmore preferably 95% or higher. The algorithm stated herein can be usedto determine the homology. Such polynucleotides are useful as probes forthe isolation or detection of DNA encoding the protein of the inventionas stated in a later example or as a primer used for amplifications.

The antisense oligonucleotide derivatives of the present invention actupon cells producing the protein of the invention by binding to the DNAor mRNA encoding the protein, inhibiting its transcription ortranslation, promoting the degradation of the mRNA, and inhibiting theexpression of the protein of the invention, thereby resulting in theinhibition of the protein's function.

An antisense oligonucleotide derivative of the present invention can bemade into an external preparation, such as a liniment or a poultice, bymixing with a suitable base material which is inactive against thederivatives.

Also, as needed, the derivatives can be formulated into tablets,powders, granules, capsules, liposome capsules, injections, solutions,nose-drops and freeze-drying agents by adding excipients, isotonicagents, solubilizers, stabilizers, preservatives, pain-killers, andsuch. These can be prepared by following usual methods.

The antisense oligonucleotide derivative is given to the patient bydirectly applying onto the ailing site or by injecting into a bloodvessel so that it will reach the site of ailment. An antisense-mountingmedium can also be used to increase durability andmembrane-permeability. Examples are, liposome, poly-L-lysine, lipid,cholesterol, lipofectin or derivatives of these.

The dosage of the antisense oligonucleotide derivative of the presentinvention can be adjusted suitably according to the patient's conditionand used in desired amounts. For example, a dose range of 0.1 to 100mg/kg, preferably 0.1 to 50 mg/kg can be administered.

The antisense oligonucleotide of the invention inhibits the expressionof the protein of the invention and is thereby useful for suppressingthe biological activity of the protein of the invention. Also,expression-inhibitors, comprising the antisense oligonucleotide of theinvention, are useful in the point that they can inhibit the biologicalactivity of the protein of the invention.

Moreover, the present invention provides a method of screening for acompound that binds to the protein of the present invention by using theprotein of the present invention. This screening method comprises thesteps of: (a) contacting the protein of the present invention or apartial peptide thereof with a subject sample, (b) detecting the bindingactivity between the protein of the present invention or the partialpeptide thereof and the subject sample, and (c) selecting a compoundthat binds to the protein of the present invention or the partialpeptide thereof.

The protein of the present invention to be used for screening may be arecombinant protein or a protein derived from the nature, or a partialpeptide thereof. Any subject sample, for example, cell extracts, cellculture supernatant, products of fermenting microorganism, extracts frommarine organism, plant extracts, purified or crude proteins, peptides,non-peptide compounds, synthetic micromolecular compounds and naturalcompounds, can be used. The protein of the present invention to becontacted with a subject sample can be, for example, a purified protein,a soluble protein, a form bound to a carrier, or a fusion protein fusedwith other proteins.

As a method of screening for proteins, for example, that bind to theprotein of the present invention using the protein of the presentinvention, many methods well known by a person skilled in the art can beused. Such a screening can be conducted by, for example,immunoprecipitation method, specifically, in the following manner. Thegene encoding the protein of the present invention is expressed inanimal cells and so on by inserting the gene to an expression vector forforeign genes, such as pSV2neo, pcDNA I, and pCD8. The promoter to beused for the expression may be any promoter that can be used commonlyand include, for example, the SV40 early promoter (Rigby in Williamson(ed.), Genetic Engineering, vol. 3. Academic Press, London, p. 83-141(1982)), the EF-1a promoter (Kim et al., Gene, 91:217-223, 1990), theCAG promoter (Niwa et al., Gene, 108:193-200, 1991), the RSV LTRpromoter (Cullen, Methods in Enzymology, 152:684-704, 1987) the SRαpromoter (Takebe et al., Mol. Cell. Biol., 8:466, 1988), the CMVimmediate early promoter (Seed et al., Proc. Natl. Acad. Sci. USA,84:3365-3369, 1987), the SV40 late promoter (Gheysen et al., J. Mol.Appl. Genet., 1:385-394, 1982), the Adenovirus late promoter (Kaufman etal., Mol. Cell. Biol., 9:946, 1989), the HSV TK promoter and so on. Theintroduction of the gene into animal cells to express a foreign gene canbe performed according to any methods, for example, the electroporationmethod (Chu et al., Nucl. Acids Res., 15:1311-1326, 1987), the calciumphosphate method (Chen et al., Mol. Cell. Biol., 7:2745-2752, 1987), theDEAE dextran method (Lopata et al., Nucl. Acids Res., 12:5707-5717,1984; Sussman et al., Mol. Cell. Biol., 4:1642-1643, 1985), theLipofectin method (Derijard, Cell, 7:1025-1037, 1994; Lamb et al.,Nature Genetics, 5:22-30, 1993; Rabindran et al., Science, 259:230-234,1993), and so on. The protein of the present invention can be expressedas a fusion protein comprising a recognition site (epitope) of amonoclonal antibody by introducing the epitope of the monoclonalantibody, whose specificity has been revealed, to the N- or C-terminusof the protein of the present invention. A commercially availableepitope-antibody system can be used (Experimental Medicine, 13:85-90,1995). Vectors which can express a fusion protein with, for example,β-galactosidase, maltose binding protein, glutathione S-transferase,green florescence protein (GFP) and so on by the use of its multiplecloning sites are commercially available.

A fusion protein prepared by introducing only small epitopes consistingof several to a dozen amino acids so as not to change the property ofthe protein of the present invention by the fusion is also reported.Epitopes, such as polyhistidine (His-tag), influenza aggregate HA, humanc-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag),E-tag (an epitope on monoclonal phage), and such, and monoclonalantibodies recognizing them can be used as the epitope-antibody systemfor screening proteins binding to the protein of the present invention(Experimental Medicine, 13:85-90, 1995).

In immunoprecipitation, an immune complex is formed by adding theseantibodies to cell lysate prepared by using an appropriate detergent.The immune complex consists of the protein of the present invention, aprotein comprising the binding ability with the protein, and anantibody. Immunoprecipitation can be also conducted by using antibodiesagainst the protein of the present invention, besides using antibodiesagainst the above epitopes. An antibody against the protein of thepresent invention can be prepared, for example, by introducing a geneencoding the protein of the present invention to an appropriate E. coliexpression vector, expressing the gene in E. coli, purifying theexpressed protein, and immunizing rabbits, mice, rats, goats, domesticfowls and such against the protein. The antibody can be also prepared byimmunizing the above animals against a synthesized partial peptide ofthe protein of the present invention.

An immune complex can be precipitated, for example by Protein ASepharose or Protein G sepharose when the antibody is a mouse IgGantibody. If the protein of the present invention is prepared as afusion protein with an epitope, such as GST, an immune complex can beformed in the same manner as in the use of the antibody against theprotein of the present invention, by using a substance specificallybinding to these epitopes, such as glutathione-Sepharose 4B.

Immunoprecipitation can be performed by following or according to, forexample, the methods in the literature (Harlow, E. and Lane, D.:Antibodies pp. 511-552, Cold Spring Harbor Laboratory publications, NewYork (1988)).

SDS-PAGE is commonly used for analysis of immunoprecipitated proteinsand the bound protein can be analyzed by the molecular weight of theprotein by using gels with an appropriate concentration. Since theprotein bound to the protein of the present invention is difficult todetect by a common staining method, such as Coomassie staining or silverstaining, the detection sensitivity for the protein can be improved byculturing cells in culture medium containing radioactive isotope,³⁵S-methionine or ³⁵S-cystein, labeling proteins in the cells, anddetecting the proteins. The target protein can be purified directly fromthe SDS-polyacrylamide gel and its sequence can be determined, when themolecular weight of a protein has been revealed.

As a method for isolating proteins binding to the protein of the presentinvention by using the protein, for example, West-Western blottinganalysis (Skolnik et al., Cell, 65:83-90, 1991) can be used.Specifically, a protein binding to the protein of the present inventioncan be obtained by preparing a cDNA library from cells, tissues, organs(for example, tissues such as ovary, testis, and placenta or culturedcells) expected to express a protein binding to the protein of thepresent invention by using a phage vector (λgt11, ZAP, etc.), expressingthe protein on LB-agarose, fixing the protein expressed on a filter,reacting the purified and labeled protein of the present invention withthe above filter, and detecting the plaques expressing proteins bound tothe protein of the present invention according to the label. The proteinof the invention may be labeled by utilizing the binding between biotinand avidin, or by utilizing an antibody that specifically binds to theprotein of the present invention, or a peptide or polypeptide (forexample, GST) that is fused to the protein of the present invention.Methods using radioisotope or fluorescence and such may be also used.

Alternatively, in another embodiment of the screening method of thepresent invention, a two-hybrid system utilizing cells may be used(“MATCHMAKER Two-Hybrid system”, “Mammalian MATCHMAKER Two-Hybrid AssayKit”, “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-HybridVector System” (Stratagene); the references “Dalton S, and Treisman R(1992) Cell 68, 597-612”, “Fields S, and Sternglanz R. Trends Genet.(1994) 10:286-292”).

In the two-hybrid system, the protein of the invention is fused to theSRF-binding region or GAL4-binding region and expressed in yeast cells.A cDNA library is prepared from cells expected to express a proteinbinding to the protein of the invention, such that the library, whenexpressed, is fused to the VP16 or GAL4 transcriptional activationregion. The cDNA library is then introduced into the above yeast cellsand the cDNA derived from the library is isolated from the positiveclones detected (when a protein binding to the protein of the inventionis expressed in yeast cells, the binding of the two activates a reportergene, making positive clones detectable). A protein encoded by the cDNAcan be prepared by introducing the cDNA isolated above to E. coli andexpressing the protein.

As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene,luciferase gene and such can be used besides HIS3 gene.

A compound binding to the protein of the present invention can bescreened using affinity chromatography. For example, the protein of theinvention may be immobilized on a carrier of an affinity column, and atest sample, containing a protein capable of binding to the protein ofthe invention is supposed to be expressed, is applied to the column. Atest sample herein may be, for example, cell extracts, cell lysates,etc. After loading the test sample, the column is washed, and proteinsbound to the protein of the invention can be prepared.

The amino acid sequence of the obtained protein is analyzed, an oligoDNA is synthesized based on the sequence, and cDNA libraries arescreened using the oligo DNA as a probe to obtain a DNA encoding theprotein.

A biosensor using the surface plasmon resonance phenomenon may be usedas a mean for detecting or quantifying the bound compound in the presentinvention. When such a biosensor is used, the interaction between theprotein of the invention and a test compound can be observed real-timeas a surface plasmon resonance signal, using only a minute amount ofprotein and without labeling (for example, BIAcore, Pharmacia).Therefore, it is possible to evaluate the binding between the protein ofthe invention and a test compound using a biosensor such as BIAcore.

The methods of screening for molecules that bind when the immobilizedprotein of the present invention is exposed to synthetic chemicalcompounds, or natural substance banks, or a random phage peptide displaylibrary, or the methods of screening using high-throughput based oncombinatorial chemistry techniques (Wrighton et al., Science (US),273:458-64, 1996; Verdine, Nature (England), 384:11-13, 1996; Hogan,Nature (England), 384:17-19, 1996) to isolate not only proteins butchemical compounds that bind to protein of the present invention(including agonist and antagonist) are well known to one skilled in theart.

A compound isolated by the screening is a candidate for drugs whichpromote or inhibit the activity of the protein of the present invention,for treating or preventing diseases attributed to, for example, thefunctional abnormality of the protein of the present invention, or cellproliferative diseases such as cancer. A compound in which a part of thestructure of the compound obtained by the present screening methodhaving the activity of binding to the protein of the present inventionis converted by addition, deletion and/or replacement, is included inthe compounds obtained by the screening method of the present invention.

Moreover the present invention provides a method for screening acompound which promotes or inhibits the activity of the protein of thepresent invention. Since the Gros1 protein of the present invention hasthe activity of inhibiting cell proliferation, a compound which promotesor inhibits this activity of Gros1 protein of the present invention canbe screened using this activity as an index.

This screening method includes the steps of: (a) culturing cells whichexpress Gros1 protein in the presence of the subject sample, (b)detecting the proliferation of the cells, and (c) selecting a compoundwhich promotes or inhibits the proliferation in comparison with theproliferation detected in the absence of the subject sample.

Any Gros1 proteins can be used for screening so long as they comprisethe activity of inhibiting cell proliferation. For example, human ormouse Gros1-L protein can be used and proteins functionally equivalentto these proteins can also be used. Gros1 proteins may be expressedendogenously or exogenously by cells.

Any subject samples, for example, cell extracts, cell culturesupernatant, products of fermenting microorganism, extracts of marineorganism, plant extracts, purified or crude proteins, peptides,non-peptide compounds, synthetic micromolecular compounds, naturalcompounds, can be used. A compound obtained by the above screening forcompounds that bind to the protein of the present invention can be alsoused as the subject compound.

The compound isolated by this screening is a candidate for agonists orantagonists of the protein of the present invention. The term “agonist”refers to molecules that activate the function of the protein of thepresent invention by binding thereto. Likewise, the term “antagonist”refers to molecules that inhibit the function of the protein of thepresent invention by binding thereto. Moreover, a compound isolated bythis screening is a candidate for compounds which inhibit the in vivointeraction of the protein of the present invention with molecules(including DNAs and proteins).

Cell proliferation can be detected, for example, by determining thespeed of cell proliferation, measuring the cell cycle and such, as wellas by measuring the colony forming activity as described in theExamples.

The compound isolated by the screening is a candidate for drugs whichpromote or inhibit the activity of the protein of the present inventionand can be applied to the treatment for diseases (for example, cancer,etc.) associated with the protein of the present invention.

Moreover, compound in which a part of the structure of the compoundpromoting or inhibiting the activity of Gros1 proteins is converted byaddition, deletion and/or replacement are also included in the compoundsobtainable by the screening method of the present invention.

When administrating the compound isolated by the method of the inventionas a pharmaceutical for humans and other mammals, such as mice, rats,guinea-pigs, rabbits, chicken, cats, dogs, sheep, pigs, cattle, monkeys,baboons, chimpanzees, the isolated compound can be directly administeredor can be formulated into a dosage form using known pharmaceuticalpreparation methods. For example, according to the need, the drugs canbe taken orally, as sugar-coated tablets, capsules, elixirs andmicrocapsules, or non-orally, in the form of injections of sterilesolutions or suspensions with water or any other pharmaceuticallyacceptable liquid. For example, the compounds can be mixed withpharmacologically acceptable carriers or medium, specifically,sterilized water, physiological saline, plant-oil, emulsifiers,suspending agents, surfactants, stabilizers, flavoring agents,excipients, vehicles, preservatives, binders and such, in a unit doseform required for generally accepted drug implementation. The amount ofactive ingredients in these preparations makes a suitable dosage withinthe indicated range acquirable.

Examples of additives that can be mixed to tablets and capsules are,binders such as gelatin, corn starch, tragacanth gum and arabic gum;excipients such as crystalline cellulose; swelling agents such as cornstarch, gelatin and alginic acid; lubricants such as magnesium stearate;sweeteners such as sucrose, lactose or saccharin; flavoring agents suchas peppermint, Gaultheria adenothrix oil and cherry. When the unitdosage form is a capsule, a liquid carrier, such as oil, can also befurther included in the above ingredients. Sterile composites forinjections can be formulated following normal drug implementations usingvehicles such as distilled water used for injections.

Physiological saline, glucose, and other isotonic liquids includingadjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodiumchloride, can be used as aqueous solutions for injections. These can beused in conjunction with suitable solubilizers, such as alcohol,specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, non-ionic surfactants, such as Polysorbate 80™ andHCO-50.

Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may beused in conjunction with benzyl benzoate or benzyl alcohol as asolubilizers and may be formulated with a buffer, such as phosphatebuffer and sodium acetate buffer; a pain-killer, such as procainehydrochloride; a stabilizer, such as benzyl alcohol, phenol; and ananti-oxidant. The prepared injection may be filled into a suitableampule.

Methods well known to one skilled in the art may be used to administerthe inventive pharmaceutical compound to patients, for example asintraarterial, intravenous, percutaneous injections and also asintranasal, transbronchial, intramuscular or oral administrations. Thedosage and method of administration vary according to the body-weightand age of a patient and the administration method; however, one skilledin the art can routinely select them. If said compound is encodable by aDNA, the DNA can be inserted into a vector for gene therapy and thevector administered to perform the therapy. The dosage and method ofadministration vary according to the body-weight, age, and symptoms of apatient but one skilled in the art can select them suitably.

For example, although there are some differences according to thesymptoms, the dose of a compound that binds with the protein of thepresent invention and regulates its activity is about 0.1 mg to about100 mg per day, preferably about 1.0 mg to about 50 mg per day and morepreferably about 1.0 mg to about 20 mg per day, when administered orallyto a normal adult (weight 60 kg).

When administering parenterally, in the form of an injection to a normaladult (weight 60 kg), although there are some differences according tothe patient, target organ, symptoms and method of administration, it isconvenient to intravenously inject a dose of about 0.01 mg to about 30mg per day, preferably about 0.1 to about 20 mg per day and morepreferably about 0.1 to about 10 mg per day. Also, in the case of otheranimals too, it is possible to administer an amount converted to 60 kgsof body-weight.

All publications and patents cited herein are incorporated by referencein their entirety.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the alignments for the mouse Gros1 sequence and the ESTsequence.

FIG. 2 shows the splicing form for cDNA of mouse Gros1.

FIG. 3 shows the splicing form for cDNA of human Gros1.

FIG. 4 shows a photograph indicating the result of Northern analysis inmouse tissues using the mouse Gros1 cDNA probe.

FIG. 5 shows photographs indicating the result of Northern analysis inhuman cells using the mouse Gros1 cDNA probe.

FIG. 6 shows a photograph indicating the result of Northern analysis inhuman tissues using the mouse Gros1 cDNA probe.

FIG. 7 shows a photograph indicating the result of Western analysis forhuman GFP-Gros1L and GFP-Gros1S expressed in COST.

FIG. 8 shows photographs indicating localization of human GFP-Gros1L andhuman GFP-Gros1S in cells.

FIG. 9 shows photographs depicting the result of Northern analysis inNIH3T3 cells to which mouse Gros1L, Gros1 mutant and Gros1 antisensewere introduced.

FIG. 10 shows the result of analysis of colony forming activity ofNIH3T3 cells to which mouse Gros1L, Gros1 mutant and Gros1 antisensewere introduced.

DETAILED DESCRIPTION

The present invention is illustrated in details by following Examples,but is not restricted to these Examples.

Example 1 Cloning and Sequence of Gros1 cDNA

It is known that the comparison of the proteins contained in the TritonX-100 insoluble factions of the plasma membrane of mouse normal cells(CMEF) and immortalized cells (NIH3T3) revealed that the protein (p33),about 30 kDa in size, present in NIH3T3 is not contained in CMEF (Wadhwaet al., Mutat. Res., 256:243-254, 1991). This protein was isolated bySDS-PAGE and anti-p33 polyclonal antibody was prepared by a standardmethod. A novel gene Gros1-L was obtained from an RS-4 cell cDNA library(Wadhwa et al., J. Biol. Chem., 268:6615-6621, 1993) by immunoscreeningusing this antibody. The sequence of this gene was novel, and nohomologous sequences were found in the DNA sequence data bank. Screeningof the human testis library (prepared based on pCMV-SPORT (GIBCO BRLCat. #10419-018); D'Alessio et al., Focus, 12:47, 1990; Kriegler, M.,1990, Gene Transfer and Expression: A Laboratory Manual, Stockton Press,New York, N.Y.; Sambrook, J. et al., 1989, Molecular Cloning: ALaboratory Manual, 2^(nd) Edition. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.; Li et al., BioTechniques, 16:722, 1994) using a ³²Plabeled mouse Gros1 probe identified two types of clones (human Gros1-Land Gros1-S). Mouse Gros1-S was identified by searching the EST datausing the nucleotide sequences of the obtained mouse Gros1 cDNAfragments and connecting overlapped clones (FIG. 1). This EST containeda 94 bp deletion in comparison with mouse Gros1-L or other ESTs, and waspredicted to generate a protein (mouse Gros1-S) shorter than thenon-deleted type (mouse Gros1-L).

SEQ ID NOs:5 and 7 show the full length nucleotide sequences of mouseGros1-L cDNA and the full length nucleotide sequence of mouse Gros1-ScDNA obtained by the cloning using EST search, respectively. Amino acidsequences deduced from these nucleotide sequences are shown in SEQ IDNOs:6 and 8, respectively. It was revealed that each of the obtainedfull-length cDNA sequences shared 85.9% homology with the novel basementmembrane-associated proteoglycan (leprecan) isolated from rat cDNA inthe DNA sequence data bank. The cDNA obtained by immunoscreening (mouseGros1-L) and the cDNA obtained by the cloning using EST search encoded amouse Gros1-S, an 85 kDa protein consisting of 747 amino acids and a61.5 kDa protein consisting of 542 amino acids (FIG. 2), respectively,having 90.9% homology with the rat leprecan in the protein data bank.Moreover, the above human 3.0 Kb clone cDNA (SEQ ID NO:1) and the cDNAof 2.7 kb clone (SEQ ID NO:3) having 83.9% homology with the mouse Gros1were revealed to have 81.9% homology with the rat leprecan in the DNAsequence data bank. The obtained 3.0 Kb clone cDNA (SEQ ID NO:1) encodedhuman Gros1-S of 41 kDa consisting of 363 amino acids (SEQ ID NO:2), andthe 2.7 Kb clone cDNA (SEQ ID NO:3) encoded human Gros1-L of 83 kDaconsisting of 736 amino acids (SEQ ID NO:4) (FIG. 3), each having 83.0%homology with the leprecan within the protein databank. Although nomatching DNA sequences were found, analysis of amino acid sequences bymotif search revealed that the amino acid sequences in mouse and humanGros1-L partially comprise the leucine zipper structure frequently foundin transcriptional factors.

Example 2 Preparation of Recombinant Gros1

The cDNA at 183-1055 within the mouse Gros1-L cDNA open reading framewas amplified by the PCR reaction (94° C. for 1 min, 55° C. for 1 min,and 72° C. for 3 min, 25 cycles) using a sense primer comprising theBamHI site (SEQ ID NO:9) and an antisense primer comprising the HindIIIsite (SEQ ID NO:10), and the obtained product was inserted into pGEM-Teasy vector (Promega) using Rapid Ligation Kit (Boehringer Mannheim).The mixture solution of E. coli JM109 competent cells (TOYOBO) and thevector were treated at 42° C. for 1 min, spread on an ampicillin plate,and cultured for 1 day, and colonies were collected for cloning. Toprepare the histidine-tagged protein, the cloned pGEM-T/Gros1 vector wascleaved at BamH I-Hind III sites, ligated with pQE30 (Qiagen), anddigested at the same restriction sites by the same manner as above, andcolonies were collected to obtain plasmids. E. coli M15 (Qiagen) wascultured until the absorbance at 580 nm reached 0.6, at which point thecells were transformed by the collected plasmids, and proteins wereproduced by inducing at 37° C. for 3 hours with 0.2 mM IPTG. Lysate ofthis E. coli was separated by SDS-PAGE method and detection wasconducted by the Western blot analysis with the histidine antibody andGros1 antibody described below. As a result, it was confirmed that a 40kDa protein was synthesized. The size of the recombinant protein was aspredicted. No signals could be detected by the Western blot analysisusing the anti-p33 polyclonal antibody in the same manner.

Example 3 Northern Blot Analysis

Northern blot analysis was conducted by purchasing a membrane on which 2μg of mRNA from various mouse, human tissues per lane were loaded(Clontech laboratories, Palo alto, CA). The gene fragment of mouseGros1-L plasmid was used as a probe. Condition for the hybridization wasas follows: “Rapid-hyb buffer” (Amersham LIFE SCIENCE) was used, andafter prehybridization at 68° C. for 30 min, the labeled probes wereadded, and the solution was incubated at 68° C. for 2 hours to performhybridization. Then washing was conducted 3 times in 2×SSC, 0.01% SDS atroom temperature for 20 min, then 3 times in 1×SSC, 0.1% SDS at 37° C.for 20 min, and twice in 1×SSC, 0.1% SDS at 50° C. for 20 min. Detectionwas performed by autoradiography. Northern blot analysis showed that the4.4 kb and 2.5 kb bands were weakly expressed in most tissues in human,except for testis, ovary, and placenta. In contrast, in testis, ovary,and placenta, very strong expression was observed (FIG. 4). Higherexpression of mRNA in human cultured cells was observed than in tissues.Moreover, in human normal cultured cells, the expression amount of the2.5 kb mRNA was nearly 10 times as high as that for the mRNA of 4.4 kb(FIG. 5). In mouse, the 3.5 kb and 2.5 kb bands were weakly expressed inmost tissues, except for brain, spleen and testis. No expression wasobserved in brain or spleen, and in testis only the 2.5 kb band wasexpressed. In testis and ovary, only the short type of Gros1 mRNAs wasdetected. It was shown that the expression dramatically disappeared atthe 11^(th) day during developmental process (FIG. 6).

Example 4 Location on the Chromosomes

The locations of the genes of the present invention were determined byusing a sense primer (SEQ ID NO:11) and an antisense primer (SEQ IDNO:12) specific to human Gros1, with a radiation hybrid panel. As aresult, they were found to be present on chromosome 1p31 in human. Inmouse, they were deduced to be present on chromosome 4.

Example 5 Preparation of Antibodies Specifically Binding to the Gros1Proteins

Antibodies against the recombinant protein deduced from the genesequence of Gros1 were prepared. Specifically, the recombinant mouseGros1-L protein with the histidine-tag prepared in Example 2 waspurified by a nickel column; rabbits were immunized to extract the serum4 times stepwise; and, finally, exsanguinations were conducted.Polyclonal antibodies were prepared by purifying this serum using theprotein A column. It was confirmed that this anti-Gros1 polyclonalantibody recognizes Gros1 protein by separating the recombinanthistidine-tagged human Gros1-L protein on the gel by SDS-PAGE, anddetecting by the Western blot analysis.

Example 6 Western Blot Analysis

Western blot analysis on the lysate of human normal lung fibroblastcell, MRC-5, with the above Gros1 polyclonal antibodies detected a bandabout 83 kDa and another about 41 kDa which were expected from the cDNAsequence. The fact that two bands were detected is consistent with thefact that two types of transcripts, one of about 4.4 and the other ofabout 2.5 kb, respectively, were detected in the Northern analysis.Interestingly, in HeLa cells, in which only the long band was detectedby Northern analysis, only the 83 kDa band was detected by the Westernanalysis. On the other hand, in NIH3T3 cells, not only the band of about85 kDa was detected by anti-Gros1 antibody, but also bands with the sizeof 61.5, 41, 34 and 32 kDa were detected. The bands of about 85 kDa and61.5 kDa correspond to mouse Gros1-L and mouse Gros1-S, respectively,and the other bands correspond presumably to proteins cleaved ormodified endogenously. In COS7 cells, 60, 40 and 34 kDa bands weredetected. The cDNAs encoding either human Gros1-L or S were insertedinto expression vectors and transfected into COS7 cells. As theexpression vector, pCMV-SPORT Vector (GIBCO BRL) used in the screeningof human testis library was used. As a result of Western blot analysisusing anti-Gros1 antibody, either an 83 kDa band or 41 kDa bandcorresponding to the cDNA sequence was detected. In COS7 cells, in whichthis plasmid encoding GFP-Gros1 fusion protein described below wastransfected, production of proteins corresponding to the sizes ofGros1-L or Gros1-S (115 kDa and 72 kDa, respectively) was confirmed byWestern blot analysis after SDS-PAGE.

Example 7 Localization of the Genes in the Cell

Two types of human Gros1 cDNA were amplified by PCR using sense (SEQ IDNO:13) and antisense (SEQ ID NO:14, 15) primers designed so as tocomprise two types of open reading frames corresponding to human Gros1-Land S. “GFPC1/7-3.0”, which expresses the fusion protein of humanGros1-S, and “GFPC1/7-2.7”, which expresses the fusion protein of humanGros1-L, were prepared by inserting these genes to the C terminal regionof GFP ORF in pEGFP-C1 (Clontech). With the usage of Tfx-50 (Promega),these plasmids encoding the GFP-Gros1 fusion protein and the controlsplasmids encoding only the GFP were transfected into COS7 cells growingon the cover glass. The cells were fixed with 4% formaldehyde, 24 hoursafter the transfection, and were washed three times with PBS. The cellswere observed with an epifluorescence Olympus BH-2 microscope. As aresult, the two types of proteins fused with different types of Gros1full-length sequences were both localized in the cytoplasm (FIGS. 7 and8).

Example 8 Proliferation Repressing Activity

The Gros1 mutant cDNA/pBluescript encoding only the 369 amino acids atthe N-terminus of mouse Gros1-L isolated by screening was cleaved withEcoRI, and ligated in the same manner as in Example 2, with SRaexpression vector (Mol. Cell. Biol., 8:466-472, 1988), and treated withthe restriction enzyme EcoRI. As a result, two clones, in sense andantisense directions, respectively, were obtained. To isolate a geneencoding the full-length mouse Gros1, EST clone AA49892A, which showedhomology with mouse Gros1, was purchased from Genome System. The ESTclone and Gros1 cDNA/pBluescript were both treated with restrictionenzymes ScaI and NotI, and the gene fragments were ligated in the samemanner as in Example 2 to obtain the mouse Gros1-L gene. This Gros1-Lgene fragment was further treated with restriction enzymes at theEcoRI-NotI site and ligated to the SRα expression vector treated withrestriction enzymes at the same site in the same manner as in Example 2.As a result, the SRa/Gros 1-L sense clone which expresses mouse Gros1-Lwas isolated.

Six G418 resistance clones were obtained by introducing the abovevectors into NIH3T3 cells and expression of Gros1 in each vector wasconfirmed by Northern blot analysis (FIG. 9). Among these, a clone insense direction with especially high expression and a clone in antisensedirection in which endogenous Gros1 transcript was rarely detected byNorthern analysis were subjected to the colony forming activity test.

500 cells of each clone were spread on a 10 cm dish, and cultured for 2weeks by replacing the medium once every three days. The cells werefixed with PBS containing 4% formaldehyde and stained with methyleneblue to count the number of colonies. The experiments were done intriplicate.

As a result, whereas colony formation was extremely delayed in clonestransfected with Gros1-L in the sense direction, the colony formation inclones transfected with Gros1-L in the antisense direction was as about5 times higher than the control (FIG. 10, Table 1). In Gros1-mutant inthe sense direction, no decrease of colony formation was observed(“defective colonies” in Table 1). From results above, Gros1 protein wasshown to have an activity to repress proliferation.

TABLE 1 Number of colonies Cloned Cell Dish 1 Dish 2 Control coloniesControl 702 197 150 Control 733 223 106 Sense colonies NIH3T3/7-7m3 S238 42 NIH3T3/7-7m3 S4 23 40 NIH3T3/7-7m3 S5 40 60 NIH3T3/7-7m3 S6 33 9NIH3T3/7-7m3 S10 39 16 Antisense colonies #723 181 186 #AS1 190 169 #AS2276 336 #AS3 398 341 #AS10 209 187 #AS12 233 254 Defective colonies #715201 215 #719 179 193 #774 156 113 #784 103 117 #738 97 80

INDUSTRIAL APPLICABILITY

The presence of non-random mutations on human chromosome 1p in manymalignant tumors was proposed by cytogenetic and molecular biologicalapproaches. These facts suggest that one or more gene mutations onchromosome 1p are important for malignant tumors. As the human Gros1gene of the present invention is present on the chromosome 1p region andhas the activity to suppress tumors, this gene may be a causative genefor these diseases. Therefore, the proteins or genes of the presentinvention, as well as a compound which promotes the activity of theproteins of the present invention can be used as useful tools forpurifying and cloning novel factors involved in cell proliferation, andfurthermore, can be used for developing pharmaceuticals for treating orpreventing various tumors.

1. An isolated nucleic acid comprising a sequence encoding a proteincomprising the amino acid sequence of SEQ ID NO:2.
 2. A vector intowhich the nucleic acid of claim 1 is inserted.
 3. An isolatedtransformant harboring the nucleic acid of claim
 1. 4. An isolatedtransformant harboring the vector of claim
 2. 5. A method for producinga polypeptide, the method comprising the steps of culturing thetransformant of claim 3 and recovering the protein from the transformantor the culture supernatant thereof.
 6. The nucleic acid of claim 1,wherein the nucleic acid comprises the coding region of the nucleotidesequence of SEQ ID NO:1.
 7. The nucleic acid of claim 1, wherein theprotein consists of the amino acid sequence of SEQ ID NO:2.
 8. Anisolated nucleic acid comprising a sequence encoding a protein thatcomprises the amino acid sequence of SEQ ID NO:2 in which ten or feweramino acids are substituted, deleted, and/or inserted.
 9. A vector intowhich the nucleic acid of claim 8 is inserted.
 10. An isolatedtransformant harboring the nucleic acid of claim
 8. 11. An isolatedtransformant harboring the vector of claim
 9. 12. A method for producinga polypeptide, the method comprising the steps of culturing thetransformant of claim 10 and recovering the protein from thetransformant or the culture supernatant thereof.
 13. The nucleic acid ofclaim 8, wherein the protein comprises the amino acid sequence of SEQ IDNO:2 in which ten or fewer amino acids are conservatively substituted.14. An isolated polynucleotide that (a) hybridizes with a nucleic acidconsisting of the nucleotide sequence of SEQ ID NO:1 or thecomplementary strand thereof and (b) comprises at least 15 nucleotides.15. An isolated nucleic acid that hybridizes under highly stringentconditions with a polynucleotide probe consisting of the complement ofnucleotides 52 to 1140 of SEQ ID NO:1, wherein said highly stringentconditions comprise washing in 2×SSC, 0.01% SDS three times at roomtemperature for 20 minutes, followed by washing in 1×SSC, 0.1% SDS threetimes at 37° C. for 20 minutes, and then washing in 1×SSC, 0.1% SDStwice at 50° C. for 20 minutes.
 16. A vector into which the nucleic acidof claim 15 is inserted.
 17. A transformant harboring the nucleic acidof claim
 15. 18. A transformant harboring the vector of claim
 16. 19. Amethod for producing a polypeptide, the method comprising the steps ofculturing the transformant of claim 17 and recovering a protein encodedby the nucleic acid from the transformant or the culture supernatantthereof.